Metamorphism in TDP-43 prion-like domain determines chaperone recognition

The RNA binding protein TDP-43 forms cytoplasmic inclusions via its C-terminal prion-like domain in several neurodegenerative diseases. Aberrant TDP-43 aggregation arises upon phase de-mixing and transitions from liquid to solid states, following still unknown structural conversions which are primed by oxidative stress and chaperone inhibition. Despite the well-established protective roles for molecular chaperones against protein aggregation pathologies, knowledge on the determinants of chaperone recognition in disease-related prions is scarce. Here we show that chaperones and co-chaperones primarily recognize the structured elements in TDP-43´s prion-like domain. Significantly, while HSP70 and HSP90 chaperones promote TDP-43 phase separation, co-chaperones from the three classes of the large human HSP40 family (namely DNAJA2, DNAJB1, DNAJB4 and DNAJC7) show strikingly different effects on TDP-43 de-mixing. Dismantling of the second helical element in TDP-43 prion-like domain by methionine sulfoxidation impacts phase separation and amyloid formation, abrogates chaperone recognition and alters phosphorylation by casein kinase-1δ. Our results show that metamorphism in the post-translationally modified TDP-43 prion-like domain encodes determinants that command mechanisms with major relevance in disease.

1. The first major claim of the manuscript is that methionine sulfoxidation greatly impairs protein propensity for LLPS. While this is possible, this central claim needs much better documentation. Data presented by the authors in this regard are largely limited to turbidimetry measurements under very limited set of conditions. Given that turbidity increase can result both from LLPS (droplet formation) as well as protein aggregation, this claim needs to be supported by technically convincing light microscopy data (preferentially fluorescence microscopy) and phase diagrams (or other types of data presentation) to determine saturation concentrations of unmodified and modified proteins under the experimental conditions used by the authors. Turbidimetry and light microscopy data shown in Fig. 1 B and C are unconvincing and likely misinterpreted, as the heterogeneous structures shown in upper panel of Fig. 1C look more like protein aggregates rather than classical liquid droplets. It is critical that LLPS and liquid-like character of the droplets are confirmed by FRAP measurements which are a standard approach in LLPS research. Also, no microscopy data are shown for the modified protein to validate the claim that this protein does not undergo LLPS.
2. Information regarding saturation concentration is also crucial to support the claim regarding the same helical propensity of the protein in the dilute and condensed phases. Fig. S6 for unmodified protein suggests rather high helical content and appears to be significantly different from those shown in other studies [e.g.,Lim et al., PLoS Biol 14, e1002338 (2016)]. Could the authors estimate the helical content from their CD spectrum and compare it with that deduced from NMR data? 4. Given that high local protein concentration within droplets greatly increases aggregation kinetics, in order to make any meaningful claim regarding the effect of methionine sulfoxidation on fibrillation propensity of TDP-43, experiments need to be done under conditions were both modified and non-modified proteins are in the dilute phase (i.e., no LLPS). This is not the case for experiments shown in Fig. 6 at a protein concentration of 100 uM, where large proportion of unmodified (but not modified) protein is likely in the condensed phase. 5. The claim that oxidized protein assembles into structurally different fibrils is based solely on a rather crude morphological data which provide no information about packing arrangements within fibril core. Thus, at the very least, the conclusions based on these morphological observations should be toned down.

The CD spectrum shown in
6. With regard to the experiments on the impact of molecular chaperones on TDP43 LLPS, these data appear somewhat preliminary. Again, more conclusive evidence should be provided that turbidity changes reflect solely LLPS and not protein aggregation. Furthermore, the observations on distinct effect of different chaperones are very descriptive in nature, with no mechanistic insight into these apparent differences.

Reviewer #3 (Remarks to the Author):
In the paper "Metamorphism in TDP-43 prion-like domain determines chaperone recognition" the authors characterized the effect of Methionine oxidation on the conformational, phase separation, fibrillation, chaperone recognition and phosphorylation properties of the prion-like domain of TDP-43 (PLD). This low complexity region comprises the stretch of residues 274-414 which is rich in Met residues and promotes the aggregation of TDP-43. Salient features of this work include: 1) Methionine oxidation prevent de-mixing of PLD by LLPS 2) It does so by preventing self-association of an hydrophobic stretch comprising residues 320-342 and by decreasing helicity at this region 3) Oxidized PLD impairs canonical fibril formation, introducing alternative aggregation pathways with morphologically distinct aggregates 4) Cytosolic chaperones, such as HSP90 and HSP70 and others, promote de-mixing of PLD by direct interactions with the hydrophobic region 5) Methionine oxidation suppresses chaperone recognition of the PLD 6) Methionine oxidation prevents phosphorylation of PLD serine residues by CK1d These in vitro results were integrated with current knowledge about the cellular behavior of TDP-43 to propose different protein outcomes upon cellular stress as observed during the onset of ALS and other dementias. The work is very interesting and provides novel insights about the role of methionine oxidation on protein conformations, phase separation and aggregation propensities. I think it would be of great interest for the general audience of Nat. Commun.
There are a few minor points that I feel should be clarified and complemented: a) The de-mixing process takes quite some amount of time. Incubations span days. The 1H-15N HSQC spectrum of de-mixed PLD ( Figure 1E and Supplementary Figures 1 and 2) show a large number of minor (unassigned) cross-peaks and some of them (between 7.6-8 ppm in 1H and around 125 ppm in 15N) could arise from new C-termini due to limited proteolysis (even in the presence of pmsf in the buffer, which has a limited lifetime in aqueous solutions). If that's the case, there might be different species separating differently. It would be helpful if the authors show an SDS-PAGE and/or a western blot of PLD and MetO PLD after de-mixing. b) For figure 3 it would be nice to include NMR data on reduced PLD309 under experimental conditions similar to those used for MetO PLD309 for direct comparisons. I realize these were previously presented by another group in Structure, 24, 1537, but including it will better highlight the effects of Met oxidation on the dynamics of PLD309.
c) The phosphorylation results are not as straightforward as the rest of the manuscript. Mainly because the authors do not present quantitative determinations of the levels of phosphorylation of the different PLDs used. Accordingly, it is difficult to say how much of the effects are due to phosphorylation or to the remnants of unmodified PLDs. Can the authors determine the levels of phosphorylation by NMR or mass spectrometry? Phosphoserine cross-peaks of 15N labeled proteins (such as those used here) have easily recognized, distinct chemical shifts that may shed some light on this. Or maybe phosphorus NMR. d) In the same line, the authors showed that phosphorylation of PLD was "drastically diminished" when PLD Met residues are oxidized and the reason for this would be that oxidation interferes with the client recognition of CK1d. This is certainly one explanation but another one, with a similar rationale, would be that oxidation of nearby Met side chains (for instance M405 and M414) might alter the epitope recognition of pS410 antibody. The antibody was raised against a reduced pSer410 substrate so Met side chain oxidation could lead to less binding to the blotted proteins and hence less amount of signal. Detection of PLD phosphorylation by NMR may also help here. If precipitation induced by phosphorylation is a problem, dissolving modified aggregates in urea and comparing with denatured unmodified references could also help.
The authors have characterized the effects of chaperones, co-chaperones and oxidation on TDP-43 aggregation and phase separation. Using complementary biophysics methods (Fluorescence, DIC, ThT and turbidity assays) and structural biology approaches (NMR, AFM, TEM), authors have shown that methionine oxidation reduces helical content of TDP-43, inhibits chaperones binding and phosphorylation, and affects phase separation and amyloid formation. Understanding the effects of protein post-translational modifications on chaperon interaction and amyloid formation is particularly important and challenging. Nevertheless some of the conclusions, drawn from NMR data, are not supported by convincing data and a major revision, including new data and quantitative analysis, is required before that the manuscript become publishable in Nature communication.
We are very grateful to Reviewer 1 for the overall favorable evaluation and for the constructive criticism. We have taken these concerns to heart and performed several additional experiments, which have led to four new figures in the revised manuscript. These new findings now provide, in our opinion, convincing data to support the conclusions advanced in the original version of the manuscript. They are described in detail below.
Major Point: NMR signals broadening is observed in the region 305-345 upon phase separation. Such effect can be due to a decrease of local flexibility (i.e. increase of order parameter S2), or self-association (increase of overall tumbling time tauC), or presence of conformational/chemical exchange in the micro-to millisecond time scale. The authors concluded that this broadening is due to intermolecular interaction and compare this signal broadening for different forms of PLD (unmodified, oxidation) or addition of chaperones. Such conclusions are not supported by adequate NMR data and analysis. The variation of the broadening can be due to various key factors such as a change in exchange process (variation of population or exchange constant), change in S2 or apparent tauC, each of these factors corresponding a different molecular event.
A rigorous quantitative investigation of order parameters, apparent tauC and exchange parameters (kex and P) using already acquired relaxation experiments and supplemented by Relaxation Dispersion experiments is a prerequisite before to draw conclusion from observed line broadening.
We are thankful to the reviewer for these comments and agree that obtaining the mentioned relaxation parameters would be very valuable towards the interpretation of the data. We have acquired additional relaxation experiments for the PLD and MetO PLD in LLPS conditions and obtained the required parameters. As shown in Supplementary Fig. 6 and Supplementary Table 2 in the revised manuscript, 15 N spin relaxation rates provide clear evidence of intermolecular associations for the PLD in LLPS conditions. In particular, we have calculated the apparent tauC from the ratio of the mean values of T1 and T2 (specifically, for the region where we observe line broadening and secondary structure propensities, covering residues 321-343) and show that the overall correlation time for the PLD doubles that for MetO, which serves as a strong indication that the PLD is assembling into larger species. Moreover, the overall higher values for ( 1 H)-15 N heteronuclearNOEs for the PLD compared to MetO PLD indicate that the PLD is more rigid. The S 2 order parameters for both proteins cannot be confidently calculated applying Lipari-Szabo ModelFree parameters due to the short and low-populated structured elements in both proteins, and to the low intensity of the heteronuclear NOES (<0.5 for the PLD). Nevertheless, we have calculated the S 2 order parameters from the chemical shifts as described by (Berjanskii and Wishart (2005) JACS 127: 14970-1) and show that the values are significantly higher for the PLD, in agreement with the loss of flexibility determined by ( 1 H)-15 N heteronuclearNOEs (Supplementary Fig. 6). For the signal broadening analysis shown in Fig.  1F, we compared the NMR signal intensity for the proteins in high vs. low concentrations. As shown in Supplementary Fig. 7, the structured elements remain identically populated in both conditions for the PLD. Therefore, the NMR signal line broadening does not emerge from a decrease in local flexibility at higher concentrations due to changes in the conformation, but rather from self-association (as indicated by the increased correlation time).
More importantly, our new 15 N CPMG relaxation dispersion data shows fast conformational exchange between species (kex= >2000 s -1 ) for the PLD, while MetO PLD shows lack of exchange. Overall, the relaxation data confirms that the PLD assembles into larger species via the double a-helix in a very dynamic and concentration-dependent fashion as indicated by NMR signal broadening (Fig. 1F), while MetO PLD remains highly flexible and largely disperse in solution even at high concentrations.
Our data largely agree with the conclusions drawn in Conicella et al. (2016) Structure, 24 1537-1549; for a very similar protein construct although under different experimental conditions. In that study, the authors concluded that TDP-43's PLD assembles into dynamic condensates via the a-helical region. In addition, it is well established that disordered proteins show significant NMR signal broadening upon intermolecular associations (for comparison, see Oroz et al. (2018) Nature Communications, 9: 4532). Based on these new results and considerations, we are confident that the NMR signal broadening observed for the PLD in LLPS conditions arises from intermolecular associations involved in LLPS.
Minor Points: 1. Top of page 9: Longer and unbranched fibrils are observed with MetO PLD suggesting alternative packing, according to authors. Such change can be due to an inhibition of secondary nucleation mechanism. The authors should address this point.
We agree with the reviewer that determining the impact of methionine sulfoxidation in the nucleation mechanisms is highly interesting. We have acquired new kinetic aggregation data (shown in new Supplementary Figure 9). In brief, the data show that the aggregation of the PLD and MetO PLD is governed by distinct mechanisms, due to their different dependence of the aggregation kinetics with the monomer concentration. In particular, the lack of a lag phase and of a concentration effect in the t1/2 suggests that the limiting step in the aggregation of the PLD are the conformational transitions towards amyloid-compatible structures (see Leonil et al. (2008) J Mol Biol 381: 1267-80). On the contrary, methionine sulfoxidation severely impairs primary nucleation, but not secondary nucleation (see Thacker et al. (2020) PNAS 117: 25272-83), as can be concluded form the significant reduction of the t1/2 upon increasing concentrations of fibril seeds (see new Supplementary Table 3). Therefore, we believe that the statement regarding the alternative packing in MetO PLD fibrils is valid. This new evidence is included in the revised version of the manuscript.
2. Fig.3: For clarity reason, comparison with data acquired with unmodified PLD should be presented, at least in supporting information.
We believe that the comparison of the 15 N spin relaxation parameters of the longer PLD and MetO PLD included in the new Supplementary Fig. 6 conveniently addresses this issue. We appreciate this comment from the reviewer and have corrected the inconsistencies. The green cylinder on top of the plots is aligned with the a-helix obtained by CYANA for MetO PLD309 (covering residues 324-332).

Fig3: error bars should be provided for panels A and C.
For clarity, we have modified the colors of the error bars (now in black). In most cases the bars are so small that are included inside the marker spheres.
5. The data reported in figure 3 clearly indicate a helical propensity populated at ca. 27%. In absence of structurally meaningful inter-residue NOE distance restraints, more caution should be taken in the analysis of structural models. With such a low number of structural restraints the structure statistic are not very relevant.
We agree with the reviewer that the structure of MetO PLD309 is hampered by the lack of meaningful NOEs, due to its high flexibility. Interpretation of NOE data for small, dynamic peptides can be complicated due to fast conformational averaging, since peptides are expected to sample a number of backbone conformations on a nanosecond time scale. The NOESY spectrum will contain crosspeaks representative of all conformations that are sufficiently populated. However, NMR parameters such as chemical shifts and coupling constants are population-weighted averaged over all conformers, and thus represent a valid data source for low-populated structured peptides (Wright PE et al. (1988) 27: 7167-75). In addition, the structure has been validated by PROCHECKNMR, showing that all the angles encompassed in the structure are present in favored regions of the Ramachandran plot (Supplementary Table 4, certified in the PDB Structure Validation Report).
6. Fig4G: The similarity of PLD NMR signals intensities in presence of different DNAJ and HSP70 do not seem to be compatible with the formation of a ternary complex. Lost in intensity is expected upon binding of HSP70. The authors should address this point.
The reviewer raises an interesting point. Because HSP70 and DNAJs mainly interact with the double a-helical region of the PLD, addition of HSP70 to a pre-formed PLD:DNAJ complex would show differences in this region if HSP70 would remove the PLD from DNAJ. Because we do not see differences in this region of the PLD in the NMR intensity plots, we conclude that the PLD remains bound to DNAJ using the a-helical region, even in the presence of HSP70. However, we see additional regions from the PLD involved in complex formation once HSP70 is added to the PLD:DNAJ complex, especially evident in the region 310-320 (where the LARKS is located, which may be relevant for chaperone recognition due to the presence of two nearby Phe residues: F313 and F316) and the region 370-380 and around 400 (probably due to the presence of F367, Y374, W385, F397 and F401). For more on the role of aromatics in chaperone client recognition see: Karagöz, et al. (2014) Cell 156: 963-74. Therefore, we conclude that NMR provides evidences of ternary complex formation. This discussion is included in the revised manuscript. Figure 9A and 9B: The authors should present the full rectangular images.

Supporting
We thank the reviewer for raising this point. The new Supplementary Figure 12  This manuscript describes the effect of methionine sulfoxidation on the liquid-liquid phase separation (LLPS) behavior and aggregation properties of the low-complexity (prion-like) domain of TDP-43. The authors propose that methionine sulfoxidation greatly impairs the capacity of the protein to undergo LLPS as well as aggregation into amyloid fibrils. It also affects the interaction of TDP-43 with a range of molecular chaperones. The general topic of this study is certainly of interest. However, there are a number of problems with experimental data and their interpretation. This, together with the lack of appropriate controls make the conclusions less than compelling.
1. The first major claim of the manuscript is that methionine sulfoxidation greatly impairs protein propensity for LLPS. While this is possible, this central claim needs much better documentation. Data presented by the authors in this regard are largely limited to turbidimetry measurements under very limited set of conditions. Given that turbidity increase can result both from LLPS (droplet formation) as well as protein aggregation, this claim needs to be supported by technically convincing light microscopy data (preferentially fluorescence microscopy) and phase diagrams (or other types of data presentation) to determine saturation concentrations of unmodified and modified proteins under the experimental conditions used by the authors. Turbidimetry and light microscopy data shown in Fig. 1 B and C are unconvincing and likely misinterpreted, as the heterogeneous structures shown in upper panel of Fig. 1C look more like protein aggregates rather than classical liquid droplets. It is critical that LLPS and liquid-like character of the droplets are confirmed by FRAP measurements which are a standard approach in LLPS research. Also, no microscopy data are shown for the modified protein to validate the claim that this protein does not undergo LLPS.
We thank the reviewer for her/his constructive critics of the manuscript. In the new version of the manuscript, we have included additional FRAP measurements, phase diagrams and protein quantification in the different phases. In particular, the FRAP experiments included in Supplementary Figure 1D show that the condensates formed by the PLD are highly fluid even after 96h of incubation (longer incubation time than the NMR relaxation experiments presented in Supplementary Figure 6). This remarkable fluidity within the condensates correlates with the high exchange determined by 15 N CPMG relaxation dispersion for the PLD in LLPS conditions (Supplementary Figure 6D). This evidence, in combination with the high resolution DIC images presented in the manuscript (Figs. 1C, 4B, 4F), allows us to conclude that turbidity arises from LLPS and not protein aggregation. In this regard, the phase diagrams shown in Supplementary Figure 1A clearly demonstrate that methionine sulfoxidation strongly impairs LLPS. Supplementary Figure 15B shows that LLPS is impeded by methionine sulfoxidation even at high protein concentrations, as monitored by DIC microscopy. Furthermore, high-resolution protein quantification shows that only ~35% of the PLD remains in the soluble fraction after the NMR experiments, while no MetO PLD is detected in the de-mixed fraction, after clarification at 20,000 g for 30 minutes (Supplementary Fig. 1C). All in all, the data demonstrate that the PLD undergoes significant LLPS in the NMR conditions forming fluid condensates, which is strongly diminished by methionine sulfoxidation.
2. Information regarding saturation concentration is also crucial to support the claim regarding the same helical propensity of the protein in the dilute and condensed phases. Figure 7 shows that the population of a-helical conformers for the PLD at 25 µM is comparable to 300 µM. Protein quantification shows that at the lower range of protein concentrations, almost all PLD protein remains in the disperse phase (~95%, Supplementary  Fig. 1C). On the other hand, the PLD at the higher range of protein concentrations undergoes significant LLPS, and only ~35% of the PLD remains soluble at 200 µM. However, MetO PLD remains largely disperse even at high protein concentrations. Therefore, we conclude that the statements included in Fig. 1G regarding disperse and de-mixed phases are valid. Fig. S6 for unmodified protein suggests rather high helical content and appears to be significantly different from those shown in other studies [e.g., Lim et al., PLoS Biol 14, e1002338 (2016)]. Could the authors estimate the helical content from their CD spectrum and compare it with that deduced from NMR data?

The CD spectrum shown in
We thank the reviewer for pointing out this difference. It must be noted that the polypeptide chains and buffers used in both studies are different. Lim et al. (2016) used the 263-414 fragment with a six His tag at the C-terminus prepared in 1 mM phosphate buffer [pH 6.8]. Moreover, 20 µM of protein were used and the helical content increased over incubation times. Here, we have used 0.3 mg/ml (aprox. 20 µM) of the His-tag cleaved 274-414 fragment freshly prepared in 5 mM potassium phosphate/10 mM NaCl [pH 6.8] and measured at 25 °C. Helix content can be estimated from the mean residue ellipticity at 222 nm ([θ]222) using the equation % a-helix = 100 ([θ]222/(−39500(1 − 2.57/n))), where n is the number of total peptide bonds (140 in our case) (Sommese et al. (2010) Prot Sci 19: 2001. This yields an a-helix content of 12%, while the NMR data (obtained averaging the Ca chemical shifts, considering 3.1 ppm Ca chemical shifts for 100 % a-helical conformers) yield around 40% for the a-helix spanning residues 321-331 and 10 % for the a-helix spanning residues 334-343. These values are in agreement with the ones reported by Conicella et al. (2016) Structure 24, 1537-49. Overall, the NMR measurements obtained at 15 °C for the PLD report an averaged 25,15% of a-helical content, which is higher than the content reported by CD at 25 °C. This difference may be attributed in part to the higher temperature of the CD measurements. In addition, it is well known (Chakrabartty et al. (1993) Biochemistry 32: 5560-5565), that aromatic residues contribute bands to the fay UV CD spectra which make the estimation of a-helical population less reliable. Since the effect of aromatic residues on 13 Ca chemical shifts is much weaker, we consider that the values measured by NMR are more accurate and precise. These differences are mentioned in the modified version of the manuscript.
4. Given that high local protein concentration within droplets greatly increases aggregation kinetics, in order to make any meaningful claim regarding the effect of methionine sulfoxidation on fibrillation propensity of TDP-43, experiments need to be done under conditions were both modified and non-modified proteins are in the dilute phase (i.e., no LLPS). This is not the case for experiments shown in Fig. 6 at a protein concentration of 100 uM, where large proportion of unmodified (but not modified) protein is likely in the condensed phase. Fig. 5C includes the aggregation kinetics obtained for 20 µM PLD and MetO PLD. In addition, Supplementary Fig. 9 includes the aggregation data for the PLD and MetO PLD at 9-20 µM protein concentration. Both proteins remain in the disperse phase at these protein concentrations (Supplementary Fig. 1C). Even at this concentration range, where both proteins remain soluble, the aggregation kinetics for MetO PLD is significantly delayed, likely because the primary nucleation is strongly affected (Supplementary Fig. 9).
5. The claim that oxidized protein assembles into structurally different fibrils is based solely on a rather crude morphological data which provide no information about packing arrangements within fibril core. Thus, at the very least, the conclusions based on these morphological observations should be toned down.
We agree with the reviewer and have qualified the claims regarding the alternative packing of the MetO PLD within the fibrils to avoid any overinterpretation of the fibrils morphological description.
6. With regard to the experiments on the impact of molecular chaperones on TDP43 LLPS, these data appear somewhat preliminary. Again, more conclusive evidence should be provided that turbidity changes reflect solely LLPS and not protein aggregation. Furthermore, the observations on distinct effect of different chaperones are very descriptive in nature, with no mechanistic insight into these apparent differences.
We agree with the reviewer on the interest of understanding the impact of chaperones on TDP-43 LLPS. Indeed, a discussion regarding the molecular basis of the differential effects observed for the different HSP40 isoforms on TDP-43 LLPS is included in the manuscript. Nonetheless, the scope of this study is to understand the structural changes triggered by methionine sulfoxidation on TDP-43 PLD, and how these changes in structure and dynamics alter LLPS, chaperone recognition, amyloid aggregation and phosphorylation. There is a remarkable interest in deciphering the differential roles of the large HSP40 family in the clearance of aggregating proteins, which may be relevant in several neurodegenerative diseases (see for instance Mok et al. (2018)  2) It does so by preventing self-association of an hydrophobic stretch comprising residues 320-342 and by decreasing helicity at this region 3) Oxidized PLD impairs canonical fibril formation, introducing alternative aggregation pathways with morphologically distinct aggregates 4) Cytosolic chaperones, such as HSP90 and HSP70 and others, promote de-mixing of PLD by direct interactions with the hydrophobic region 5) Methionine oxidation suppresses chaperone recognition of the PLD 6) Methionine oxidation prevents phosphorylation of PLD serine residues by CK1d These in vitro results were integrated with current knowledge about the cellular behavior of TDP-43 to propose different protein outcomes upon cellular stress as observed during the onset of ALS and other dementias. The work is very interesting and provides novel insights about the role of methionine oxidation on protein conformations, phase separation and aggregation propensities. I think it would be of great interest for the general audience of Nat. Commun.
We are thankful to the reviewer for her/his very positive comments.
There are a few minor points that I feel should be clarified and complemented: a) The de-mixing process takes quite some amount of time. Incubations span days. The 1H-15N HSQC spectrum of de-mixed PLD ( Figure 1E and Supplementary Figures 1 and 2) show a large number of minor (unassigned) cross-peaks and some of them (between 7.6-8 ppm in 1H and around 125 ppm in 15N) could arise from new C-termini due to limited proteolysis (even in the presence of pmsf in the buffer, which has a limited lifetime in aqueous solutions). If that's the case, there might be different species separating differently. It would be helpful if the authors show an SDS-PAGE and/or a western blot of PLD and MetO PLD after de-mixing. New Supplementary Figure 1C shows an SDS-PAGE containing 200 µM PLD and MetO PLD samples after the NMR experiments. New Supplementary Figs. 17, 18 show western blots containing 20 µM PLD and MetO PLD after the incubation for 24-120 h at 37 °C. No significant degradation products were visible. b) For figure 3 it would be nice to include NMR data on reduced PLD309 under experimental conditions similar to those used for MetO PLD309 for direct comparisons. I realize these were previously presented by another group in Structure, 24, 1537, but including it will better highlight the effects of Met oxidation on the dynamics of PLD309.
We agree with the reviewer in the interest of comparing the relaxation parameters of PLD and MetO PLD. New Supplementary Figure 6 shows the 15 N spin relaxation parameters for PLD and MetO PLD. We believe that the differences observed in this data are significant enough for a valid comparison and highlight the effects of methionine sulfoxidation on the dynamics of the PLD: MetO PLD remains highly flexible even at high concentrations, while PLD assembles into larger species via the double a-helical motif.
c) The phosphorylation results are not as straightforward as the rest of the manuscript. Mainly because the authors do not present quantitative determinations of the levels of phosphorylation of the different PLDs used. Accordingly, it is difficult to say how much of the effects are due to phosphorylation or to the remnants of unmodified PLDs. Can the authors determine the levels of phosphorylation by NMR or mass spectrometry? Phospho-serine cross-peaks of 15N labeled proteins (such as those used here) have easily recognized, distinct chemical shifts that may shed some light on this. Or maybe phosphorus NMR.
NMR has proven a very powerful tool to determine phosphorylation rates in IDPs (see Schwalbe et al. (2013) Biochemistry 52: 9068-9079). Unfortunately, CK1d phosphorylation promotes the fast aggregation of the PLD, and no phosphoserine moieties are detectable in the NMR spectra. This fast aggregation will impede any quantitative determination by solution state NMR. Yet, we observed that methionine sulfoxidation impaired CK1d phosphorylation, which is evident from the NMR and blotting data. d) In the same line, the authors showed that phosphorylation of PLD was "drastically diminished" when PLD Met residues are oxidized and the reason for this would be that oxidation interferes with the client recognition of CK1d. This is certainly one explanation but another one, with a similar rationale, would be that oxidation of nearby Met side chains (for instance M405 and M414) might alter the epitope recognition of pS410 antibody. The antibody was raised against a reduced pSer410 substrate so Met side chain oxidation could lead to less binding to the blotted proteins and hence less amount of signal. Detection of PLD phosphorylation by NMR may also help here. If precipitation induced by phosphorylation is a problem, dissolving modified aggregates in urea and comparing with denatured unmodified references could also help.
The reviewer raises a very interesting point. We have now performed additional experiments to determine if methionine sulfoxidation impacts the kinetics of CK1d phosphorylation. As shown in Supplementary Fig. 18, the PLD is phosphorylated by CK1d even in the monomeric state (20 µM protein, no incubation), and the phosphoPLD aggregates are dissolved in the SDS-PAGE. However, no monomeric MetO PLD is phosphorylated and only large aggregates (SDS-PAGE resistant) formed after long incubation times are phosphorylated. Therefore, methionine sulfoxidation impedes CK1d phosphorylation in the monomer, but not in the fibrillar aggregate. This evidence additionaly validates the detection of phosphMetO PLD by the pS410 antibody.